The present invention relates to plasmids and the use of such plasmids in the construction of eukaryotic viral vectors and the like.
Because of their large size, many recombinant eukaryotic viruses are produced via homologous recombination. Conventionally, this homologous recombination has taken place in a host eukaryotic cell permissive for the growth of the recombinant virus (see, e.g., Berkner, BioTechniques, 6, 616-628 (1988)). Homologous recombination in eukaryotic cells, however, has at least two major drawbacks. The process is time consuming, and many preferred recombinant eukaryotic viral constructions have a selective disadvantage relative to the predecessor eukaryotic viruses from which they are obtained. Therefore, if a skilled artisan attempts to create a new recombinant virus through the lengthy process of homologous recombination in a eukaryotic cell and fails to create the desired virus, that artisan will not be able to readily distinguish between the need to modify the construction technique and the possibility that the virus which is sought is not viable in that cell. Accordingly, there is a need for new methods of generating eukaryotic gene transfer vectors.
Previous improvements in the generation of gene transfer vectors have included the use of yeast-based systems (Ketner et al., Proc. Nat""l. Acad. Sci. (USA), 91, 6186-6190 (1994)), plasmid-based systems (Chartier et al., J. Virol., 70, 4805-4810 (1996); International Patent Application WO 96/25506 (Crouzet et al.), and cosmid-based systems (Miyake et al., Proc. Nat""l. Acad. Sci. (USA), 93, 1320-1324 (1996)).
The system developed by Chartier et al. includes a plasmid bearing a complete adenoviral vector genome flanked by Pac I restriction sites, which do not normally occur in the adenoviral genome. In the Chartier et al. method, adenoviral sequences contained on a linear DNA are homologously recombined in vivo into a linearized vector comprising a left and right ITR. The resultant vector is propagated, isolated, and digested with Pac I, and the restriction fragment bearing the adenoviral vector is transfected into a mammalian cell that is permissive for replication of the virus. The Chartier et a1. method provides relatively pure sources of adenoviral vector sequences. Unfortunately, the Chartier et al. method lacks a powerful, positive means of self-selecting the desired products and lacks flexibility in the way novel vectors can be produced.
International Patent Application WO 96/25506 (the ""506 application, Crouzet et al.) discloses the use of a two plasmid system with selective pressure mechanisms that enable the production of plasmids comprising adenoviral vectors flanked by unique restriction sites. Compared to other prior art methods, the ""506 method is in some ways better suited for the production of eukaryotic viral vectors. For example, the ""506 method does not require that two homologous recombination events occur simultaneously or virtually simultaneously (as does the Chartier et al. method). This temporal separation of the recombination events results in an increased opportunity to obtain both recombination steps in the vector production process. However, in order to reduce the xe2x80x9cbackgroundxe2x80x9d of undesired homologous recombination products, the ""506 system requires and employs a specialized bacterial strain. This specialized strain contains a DNA polymerase defect such that normal plasmid origins of replication will not function in the strain. However, only one such strain is described in ""506. Use of the ""506 method without the specialized strain and modified origin of replication leaves the skilled artisan with a difficult screening task, which is so substantial as to negate its improvements over the Chartier et al. method. Thus, the ""506 method lacks flexibility.
Systems described in the prior art can expedite the production of new recombinant eukaryotic viruses; however, additional flexibility and selection pressures are desired. Accordingly, the present invention provides an efficient and flexible method of preparing eukaryotic viral vectors, as well as vectors useful in practicing these methods. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a dual selection cassette (DSC) comprising first and second DNA segments having homology to a eukaryotic viral vector, and positive and negative selection genes, each operably linked to their own promoter. The present invention also provides a plasmid, pN/P, comprising an independent positive selection marker gene, origin of replication, a dual selection cassette, and at least one of the following: one or more unique restriction enzyme sites (URES), one or more flanking site-directed homologous recombination sites, or a phage packaging site. The dual selection cassette and pN/P plasmid can be used to produce eukaryotic gene transfer vectors without requiring temporally-linked double recombination events or the use of specialized bacterial strains that allow the replication of plasmids comprising conditional origins of replication. This method usefully increases the ratio of desired to undesired plasmid and vector constructs. The present invention also provides a method for construction of a library comprising or consisting of a multiplicity of the present inventive vectors comprising a multiplicity of genetic elements that may be the same or different and are assembled simultaneously in a single reaction. The features of these nucleic acids and this method allow the generation and use of eukaryotic expression libraries in the study of functional genomics and the like.
The invention may best be understood with reference to the accompanying drawings and in the following detailed description of the preferred embodiments.